ES2726804T3 - Production method of retinal pigment epithelial cell sheet - Google Patents

Abstract

A method for producing a cell sheet composed of retinal pigment epithelial cells, a tight junction formed between retinal pigment epithelial cells, and a basement membrane formed on a contact face with the collagen gel, comprising the following steps (1 ) sowing and culturing retinal pigment epithelial cells on a collagen gel to form a cell sheet composed of retinal pigment epithelial cells, the tight junction formed between retinal pigment epithelial cells, and the basement membrane formed on a contact face with the collagen gel, and (2) degrade the collagen gel with collagenase to detach the cell sheet composed of retinal pigment epithelial cells, the tight junction formed between the retinal pigment epithelial cells, and the basement membrane formed in a contact face with collagen gel.

Description

DESCRIPTION

Production method of retinal pigment epithelial cell sheet

Technical field

The present invention relates to a method of producing a cell sheet without using an artificial membrane, which comprises sowing retinal pigment epithelial cells on a collagen gel. The present invention also relates to a cell sheet for transplantation, which comprises a cell layer formed from retinal pigment epithelial cells and a basement membrane.

Prior art

At present, age-related macular degeneration (AMD) is one of the main diseases causing legal blindness in advanced countries, and is mainly observed in people over 50 years. Age-related macular degeneration is caused by changes in the macula associated with age, and is roughly divided into an exudative and an atrophic form. The exudative form of age-related macular degeneration is a disease associated with the development of neovascular vessels in the choroid, in the macula of elderly people, followed by bleeding and exudative lesion under the retinal pigment epithelium or retina, and finally, formation of scar tissue. The atrophic form of age-related macular degeneration is a disease accompanied by atrophy of the macular area and drusen accumulation. A precursor lesion that leads to macular degeneration associated with exudative and atrophic age is sometimes referred to particularly as macular degeneration associated with early ages and this lesion is also considered a pathology of macular degeneration associated with age.

For the treatment of age-related macular degeneration when it is a mild exudative form, surgical therapy such as photodynamic therapy, laser photocoagulation, neovascular vessel removal and the like, or a treatment method that points to regression and neovascular vessel extraction by pharmacological therapy such as administration of a vascular endothelial growth factor inhibitor (VEGF) involved in angiogenesis and the like. However, the aforementioned means cannot provide therapeutic effectiveness for the exudative or atrophic form that has progressed towards advanced atrophy or lack of retinal pigment epithelium (RPE). In such a case, an effective treatment method is the transplantation of retinal pigment epithelial cells or the retinal pigment epithelium to the deficient site under the retina.

Several methods of treatment have been tested by transplanting retinal pigment epithelial cells. It has been reported that transplantation of retinal pigment epithelial cells obtained from human fetuses in the form of a cell suspension shows a poor graft of the transplanted cells. In cases where a retinal pigment epithelial cell sheet that also contains Bruch's membrane and choroid detached as a corpse eye sheet was transplanted, post-surgical rejection occurred and a sufficient treatment effect was not obtained (non-patent document one). In addition, it has been pointed out that the method used by a corpse eye includes an ethical problem. On the other hand, there has been a case of improved vision of a patient with age-related macular degeneration, where a method was used that includes collecting normal retinal pigment epithelial cells and choroid of the patient and transplanting them to the site Poor under the macula. However, this method implies an extremely high burden for patients and an extremely high risk of surgery. Although attempts have been made to use the patient's own iris pigment epithelial cells collected from the patient with age-related macular degeneration for transplantation instead of retinal pigment epithelial cells, the upper limit of final vision is low and has not been obtained a sufficient effect, and even more, the burden and risk of using the patient's own cells are high (non-patent document 2). Therefore, conventional treatment methods using cells collected from a corpse eye or the patient's own cells are not practical in terms of ethics, safety, effects and the like, and a truly usable retinal pigment epithelial cell has been desired. for a transplant treatment. As one of the methods of producing a retinal pigment epithelial cell sheet, there can be mentioned a method that includes coating a cell culture substrate with an extracellular matrix and the like, and culturing retinal pigment epithelial cells thereon to form a sheet . For example, a cell sheet can be formed by culturing retinal pigment epithelial cells on a fibronectin-coated material, but the formation of a basement membrane is not disclosed, nor is a method described to extract a sheet from a sheet. container in which the sheet was formed (non-patent document 3). Given such problems, several laboratories have developed a method to form, in advance, a retinal pigment epithelial cell sheet using an artificial membrane instead of a basement membrane. However, it is feared that an artificial membrane is a disorder for transplant grafting and functional maintenance of the body. Although an artificial membrane with a less biological response has also been developed, it is not suitable for transplantation because it problematically induces inflammation and rejection associated with it, which are caused by differences with the basement membrane produced by the cell itself in the composition , properties, rigidity and the like. Moreover, in a conventional manner, a method for culturing a retinal pigment epithelial cell sheet having polarity is known. However, this is only for use of an experimental model in which the retinal pigment epithelial cells and the immortalized line derived from a living organism are converted into a sheet in a container and used directly, and the actual recovery of a sheet of epithelial cells from still has not been possible. retinal pigment provided with a basement membrane as in living organisms from a container (non-patent document 4). There is a method to obtain a retinal pigment epithelial cell sheet by exfoliation of retinal pigment epithelial cells grown directly on a culture plate, without using an extracellular matrix and the like. Such a method to detach the cells by exfoliation cannot stabilize the shape and size of the sheet, the basement membrane and the like, causes a lot of damage to the epithelial cells of retinal pigment by exfoliation, and causes, in most cases, the disintegration of the shape of the sheet and scattered cells due to exfoliation, thus failing to provide a sheet of cells usable for transplantation and the like (non-patent document 5). In addition, although it has been disclosed the coating of a cell culture substrate with collagen and the culture of the cells on the collagen, human retinal pigment epithelial cells are not used.

Moreover, to increase the gel strength, a crosslinking agent needs to be added separately, which puts the method in a position far from being convenient and fast (patent document 1). In the current situation, the production of a retinal pigment epithelial cell sheet, therapeutically effective for retinal diseases such as age-related macular degeneration and the like, and convenient and stable to prepare is still problematic.

Non-patent document 6 describes the transplantation of retinal pigment epithelial cell sheets obtained by tissue engineering in a rabbit model.

List of documents

Patent documents

Patent document 1: JP-A-2005-261292

Non-patent documents

Non-patent document 1: Ophtalmic Surg. 1991 Feb; 22 (2): 102-8.

Non-patent document 2: Tohoku J Ex Med. 1999 Dec; 189 (4): 295-305.

Non-patent document 3: Nat. Protoc., 4 (5), 2009, 662-673.

Non-patent document 4: Invest. Ophtalmol Vis. Sci., 47, 2006, 3612-3624.

Non-patent document 5: Invest. Ophtalmol Vis. Sci., 36 (2), 195, 381-390.

Non-patent document 6: Biomaterials., 30 (5), 2009, 797-803.

Brief Description of the Invention

Problems solved by the invention

The problem of the present invention is to develop a new method for producing a retinal pigment epithelial cell sheet conveniently and stably without using an artificial membrane, and providing patients with a disease associated with the lack of retinal pigment epithelium, such as age-related macular degeneration and the like, a retinal pigment epithelial cell sheet for transplantation, which shows a high graft rate and superior function.

Means to solve the problems

The present inventors produced a cell sheet by forming a layer of collagen gel on a cell culture substrate, and planting and culturing retinal pigment epithelial cells on it. A cell sheet obtained by such a method maintains a basement membrane between the collagen gel and the retinal pigment epithelial cell sheet, has the ability to secrete cytokine and adhesion between cells similar to that of retinal pigment epithelial cells in vivo, and It allows easy detachment of the retinal pigment epithelial cell sheet from the cell culture substrate while maintaining the basement membrane by collagen gel breakdown with collagenase. In addition, the cells that constitute the cell sheet maintained the expression of a specific marker of retinal pigment epithelial cells. The present inventors have conducted intensive studies and completed the present invention from these findings. Accordingly, the present invention provides

[1] a method for producing a cell sheet composed of retinal pigment epithelial cells, a tight junction formed between retinal pigment epithelial cells, and a basement membrane formed on a contact face with the collagen gel, comprising the following stages

(1) sow and grow retinal pigment epithelial cells on a collagen gel to form a sheet cell composed of retinal pigment epithelial cells, the tight junction formed between the retinal pigment epithelial cells and the basement membrane formed on a contact face with the collagen gel, and (2) degrade the collagen gel with collagenase to release the cell sheet composed of retinal pigment epithelial cells, the tight junction formed between the retinal pigment epithelial cells and the basement membrane formed on a contact face with the collagen gel;

[2] the method of production of the cell sheet of [1] mentioned above, wherein retinal pigment epithelial cells are cells obtained by inducing differentiation of stem cells or progenitor cells;

[3] the method of production of the cell sheet of [2] mentioned above, wherein the stem cells are ES cells or iPS cells;

[4] the method of production of the cell sheet of [1] mentioned above, wherein the concentration of collagen in the collagen gel is 0.1% -0.5%;

[5] the method of producing the cell sheet of any of [1] to [4] mentioned above, further comprising the following step (3)

(3) confirm the presence or absence of a basement membrane on the contact surface between the detached cell sheet and the collagen gel.

Effect of the invention

In accordance with the present invention, a retinal pigment epithelial cell sheet having a constitution having a basement membrane that appears on a surface, without using an artificial membrane, can be prepared easily and stably. Because the cell sheet of the present invention is superior in graft rate and functionality, and is extremely useful for transplantation, a sheet composed of a basal membrane and retinal pigment epithelial cells can be produced and applied to patients with ophthalmic diseases , such as patients with age-related macular degeneration and the like by transplantation. In particular, when the cell to be used for culture is a retinal pigment epithelial cell derived from an iPS cell, transplant rejection can be avoided by using the patient's own cells as a source.

Brief description of the drawings

Fig. 1 shows the results of the cytokine secretory capacity test of the retinal pigment epithelial cell sheet.

Description of the realizations

The present invention is explained in detail below.

The present invention provides a method of producing a cell sheet composed of retinal pigment epithelial cells, a tight junction formed between retinal pigment epithelial cells, and a basement membrane formed on a contact face with the collagen gel, comprising The following stages: (1) sow and cultivate retinal pigment epithelial cells on a collagen gel to form a cell sheet composed of retinal pigment epithelial cells, the tight junction formed between the retinal pigment epithelial cells and the formed basement membrane on a contact face with the collagen gel, and (2) degrade the collagen gel with collagenase to detach the cell sheet composed of retinal pigment epithelial cells, the tight junction formed between the epithelial cells of the retinal pigment and the membrane Basal formed on a contact face with the collagen gel.

Although the retinal pigment epithelial cell to be sown in step (1) can be a cell obtained from any mammal, as long as it is obtained from a mammal (for example, human, monkey, mouse, rat, dog, bovid, horse, pig, sheep, goat, cat, rabbit, hamster, guinea pig, etc.), is preferably a cell obtained from a human being.

The retinal pigment epithelial cell to be seeded may be a primary cell collected directly from an eyeball, or a cell after several passes. Primary retinal pigment epithelial cells can be isolated by a known method. For example, in the case of retinal pigment epithelial cells obtained from an eyeball, a corpse eyeball is isolated, quickly divided into the equatorial segment, the vitreous body and the retina are removed and treated with collagenase, hyaluronidase and the like as needed, the cells are collected by scraping with a cell scraper, or treatment in EDTA or trypsin solution to release the cells of the Bruch membrane, they are allowed to be in culture medium to induce adhesion to the plaque. culture and growth, and the cells grown in the required number are properly passed with a trypsin treatment etc. to ensure the number of cells.

Additionally, these cells can also be the cells obtained by inducing differentiation of undifferentiated pluripotent stem cells, such as embryonic stem cells (ES cell), induced pluripotent stem cells (iPS cell) and the like, inclined somatic stem cells such as cells mother neurals and the like, or progenitor cells including neural progenitor cells and retinal progenitor cells. In addition, as a stem cell, the target cell can be prepared by inducing differentiation of induced pluripotent stem cells (iPS cells) disclosed in recent years. An iPS cell is an induced stem cell obtained from a somatic cell that has properties equivalent to those of an ES cell, which can be produced by introducing a particular nuclear reprogramming substance (nucleic acid, protein, low molecular weight compound, etc. .) in a somatic cell [Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006); Takahashi, K. et al., Cell, 131: 861-872 (2007)]. The conditions and means used for the differentiation of the aforementioned stem cell in the object differentiated cell may be conventionally known conditions and means, or may be appropriately determined by the person skilled in the art. In the present invention, preferably a cell obtained by inducing differentiation of stem cells or progenitor cells, preferably pluripotent stem cells, is used as the epithelial cell of retinal pigment to be used for the cell sheet, since an epithelial cell of retinal pigment at an appropriate maturation stage, and in particular, comparatively immature retinal pigment epithelial cells can be prepared and a cell sheet can be advantageously formed. In addition, when the cell sheet to be produced by the present invention is for transplantation, the use of an iPS cell is preferable since a cell sheet obtained using a somatic cell of the subject, who receives transplantation, as a source of non-iPS cell It has antigenicity against the subject. When a stem cell is induced to differentiate, for example the human ES cell or pluripotent stem cell, such as the iPS cell and the like, it is cultured in an ES differentiation medium to which Wnt antagonist has been added, such as Dkk-1, CKI-7 and the like, and Nodal antagonist such as Lefty A, SB-431542 and the like. When grown for a given period, Rx, Pax6 and Mitf are expressed, which are markers of retinal progenitor cells, and human retinal pigment epithelial cells can be obtained by morphological observation with an optical microscope, confirming cells that have a polygonal shape and pigment [Neuroscience Letters 2009 Jul 24458 (3) 126-31, Journal of Cell Science 2009 Sep 1122 (Pt 17) 3169-79].

The retinal pigment epithelial cells of the present invention are cultured by seeding on a collagen gel. The collagen used for the collagen gel can be any as long as it is obtained from a mammal (for example, human, monkey, mouse, rat, dog, bovine, horse, pig, sheep, goat, cat, rabbit, hamster, guinea pig, etc.) and used, for example, collagen obtained from humans or pigs. Examples of tissue obtained from collagen include tendon, skin and the like. Although the type of collagen can be any, one other than the collagen constituting the human basement membrane is preferred, and one that is not type IV collagen is specifically preferred. Of these, type I collagen is preferably used. Although a collagen gel can be produced by, for example, a conventionally known production method, a gel composed of a network of collagen fibers inducing collagen fibrogenesis is produced in the present invention. , as described in the Example mentioned below. As fibrotic collagen has a combination of strength and flexibility, it is easy to handle, shows good maintenance of cell proliferation and cell differentiation, and is preferable as a collagen gel for use in the present invention. In addition, it is required that the collagen to be used in the present invention keep the cells, which are seeded in the collagen gel, on the gel surface without allowing them to sink into the gel layer. Therefore, as a collagen one is preferred where the gel has the necessary strength for cell proliferation and, for example, a collagen having a large amount of intermolecular cross-linking is preferable. As such collagen, collagen obtained from tendon can be mentioned.

Although the collagen concentration of the aforementioned collagen gel can be in any range as long as it can give a gel with sufficient strength to allow grafting and growth of retinal pigment epithelial cells, and which satisfies the solubility that facilitates degradation. by collagenase, the viscosity that allows easy handling and the like is preferably 0.1% (w / v) - 0.5% (w / v), more preferably 0.2% (w / v) - 0.3 % (p / v). When the collagen concentration of the collagen gel is less than 0.1% (w / v), the strength of the collagen gel becomes insufficient, and therefore, the colonization rate and the cell proliferation rate of the epithelial cells of retinal pigment. When the collagen concentration of the collagen gel exceeds 0.5% (w / v), the time of a collagenase treatment to degrade the collagen gel is lengthened, which is feared to exert an adverse influence on the cells.

Although the volume of a mixed solution of collagen gel used for the production of the aforementioned collagen gel varies depending on the culture area and form of a culture substrate to be used for cell culture, it is preferably about 100 pl - about 250 pl, more preferably about 150 pl - about 200 pl, per unit area (cm2). When the amount of collagen gel mixed solution is too small, a layer of collagen gel is formed that has a thin central part due to the influence of a surface tension applied to the gel surface, and the sheet tends to be damaged during the cutting of the cell sheet, since the cells come into direct contact with a culture substrate when the retinal pigment epithelial cells are cultured. When the amount of mixed collagen gel solution is in excess, a thick collagen gel layer is formed on a culture substrate, which relatively reduces the amount of the culture medium, and therefore, the maintenance of the culture is not It is easy to perform, collagenase treatment takes time, and damage to the cell sheet is feared.

In step (1), a cell sheet can be produced by seeding and culturing the aforementioned retinal pigment epithelial cells on the collagen gel of a cell culture substrate. The cell culture substrate in the present invention is particularly limited as long as it is for cell culture. Examples thereof include culture vessels that have a porous membrane, such as a Transwell membrane and the like, a flask, a tissue culture flask, a plate, a petri dish, a tissue culture plate, a multi-plate , a microplate, a microwell plate, a multiplate plate, a multiwell plate, a camera slide, a tube, a tray, a culture bag or a rolling bottle. Culture vessels that have a porous membrane are preferable, because a collagenase treatment and a cutting operation of the cell sheet are conveniently performed. For example, a commercially available Transwell membrane is preferably used. Examples of the cell culture substrate material herein include, but are not limited to, inorganic materials, such as metal, glass, ceramic, silicon and the like materials, organic materials represented by elastomers, plastics (eg, resin Polyester, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluoro-resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melanin resin, epoxy resin, chloride resin vinyl).

The number of retinal pigment epithelial cells to be sown can be in any range as long as the cell density is capable of forming a cell sheet. However, when the cell density is too low, the cell form is poor, the cultivation time before reaching confluence is long, and in addition, the time necessary for cell maturation and coloration is long. Similarly, when the cell density is too high, cell proliferation is suppressed, the culture time before reaching confluence tends to be long, and the cells can die from being overmastered. Therefore, the density of the cells to be sown is preferably approximately 4.5x104 cells / cm2 - approximately 8.5x105 cells / cm2, more preferably approximately 8.5x104 cells / cm2 - approximately 8.5x105 cells / cm2, very preferably 4.5x105 cells / cm2.

A population of monolayer cells (cell sheet) composed of retinal pigment epithelial cells can be formed by culturing retinal pigment epithelial cells seeded on collagen gel in a culture medium. A culture medium can be used without particular limitation as long as it is a cell culture medium generally used in the relevant field. For example, a basal medium described in "Japan tissue culture conference ed., Technique of Tissue Culture 3rd edition", page 581, published by Asakura Shoten, may be used, such as F-10 medium, F12 medium, MEM, BME medium, DMEM , aMEM, medium IMD; ES medium, DM-160 medium, Fisher medium, WE medium, RPMI1640 medium and the like. In addition, serum (fetal bovine serum, etc.), various growth factors (EGF, FGF, ^h G ^f , PDGF etc.), antibiotics, amino acids and the like to the basal medium can be added. The pH of the medium is preferably from about 6 to about 8. As for the culture, for example, a primary culture is generally carried out at a temperature of about 30 - about 40 ° C for about 15 - about 60 h until the epithelial cells of retinal pigment become confluent. Subsequently, a secondary culture is performed for approximately 1 week - approximately 2 months while the medium is changed, after which the culture is performed while aerating and stirring if necessary until the formation of a cell sheet. The cells that constitute the cell sheet obtained by such culture are maintained as retinal pigment epithelial cells. The maintenance of the cells as retinal pigment epithelial cells can be confirmed by detecting BEST1, RPE65, MERTK, CRALBP or the like as a specific differentiation marker.

Since the cell sheet formed in step (1) is adhered to the collagen gel, for example, when used directly for transplantation and the like, it is feared that the collagen gel will prevent grafting in a transplant recipient. If the collagen gel can be separated in advance, it can lead to the resolution of such a problem. In step (2) of the present invention, the collagen gel that adheres to the cell sheet formed in step (1) is degraded by collagenase. The person skilled in the art can select the appropriate collagenase according to the kind of collagen used to prepare the collagen gel. Although the collagenase to be used for degradation of the collagen gel is not particularly limited as long as it has an activity to digest the collagen gel, one that does not readily degrade the collagen constituting the human basement membrane is preferred (for example , type IV collagen). For example, collagenase obtained from a microorganism induced by Clostridium (Clostridum histolyticum) or Streptomyces (Streptomyces parvulus), which are commercially available, are safe and have high enzymatic activity can be used.

As the aforementioned collagenase activity, the specific activity in relation to the weight of collagen in the collagen gel is more important than the activity per unit of collagenase weight and that the activity per unit volume of an aqueous collagenase solution. The specific activity of the collagenase to be used to dissolve the collagen gel (collagenase activity / collagen weight) is preferably not less than 0.1 U / mg. When the specific activity of collagenase is less than 0.1 U / mg, the dissolution of the collagen gel may take longer than desirable or the gel may dissolve less than desirable. More preferably it is in the range of 0.1-10,000 U / mg, and even more preferably in the range of 1-3,000 U / mg.

In the method of production of the cell sheet of the present invention, the method of action of collagenase on collagen gel is not particularly limited. A collagenase solution prepared using a medium or an isotonic solution with pH regulating capacity as solvent can be added to the medium, or a collagen gel attached to cells detached from a cell culture plate can be immersed in the solution of collagenase mentioned above. Since a Transwell membrane is used as a cell culture substrate in the present invention, a layer of collagen gel may be exposed upon retrieving an insert and separating the membrane at the bottom of the insert, and the exposed collagen gel is preferably directly submerged. in the collagenase solution mentioned above.

In the method of producing the cell sheet of the present invention, the time to dissolve the collagen gel by collagenase is not particularly limited. When the collagenase action time is too long, cellular functions such as adhesion capacity, proliferative capacity and the like can not be degraded. Although the time of dissolution by collagenase may vary depending on the specific activity of the collagenase, temperature, the shape of the collagen gel, the method of treatment with collagenase and the like, it is generally 15 min - 60 min. Collagenase treatment can be a one-time treatment or several times.

The temperature during the treatment of collagen gel by collagenase in the method of producing the cell sheet of the present invention is preferably set within the range of 10-42 ° C, more preferably 30-40 ° C, even more preferably 36- 38 ° C, since the fluence of the cell cytoplasm generally decreases and metabolism decreases when the temperature within living organisms becomes no more than 10 ° C (approximately 30 ° C in humans), the protein is denatured and Cellular function decreases when the temperature exceeds 42 ° C, and the optimum collagenase temperature is mainly 37 ° C and a temperature below this level prolongs the dissolution time.

In the cell sheet production method of the present invention, when the collagen gel solution progresses, the cell sheet is gradually detached from the gel, and finally released into the collagenase solution. To recover the cell sheet, the cell sheet can be mechanically separated from the remaining gel, or it can be recovered after complete dissolution of the gel. Although the mechanical separation shortens the recovery time of the cell sheet, as the cell sheet can be destroyed, it is preferably recovered after complete dissolution of the gel.

Although the recovered cell sheet as mentioned above can be used directly for various applications, such as residual collagenase can inhibit the adhesiveness between the cell sheets or the adhesiveness to a tissue, preferably it is washed with a medium or an isotonic solution with regulatory capacity. of pH. The temperature during cleaning can be determined according to the collagen gel dissolution treatment by collagenase. To sufficiently remove residual collagenase, the term is preferably washed one or more times with an isotonic medium or solution having a pH regulating capacity. In the cell sheet obtained by the method of the present invention, the cytokine specific for a retinal pigment epithelial cell is secreted with a polarity similar to that found in living organisms, and transepithelial electrical resistance (TER), which is a close adhesion binding index between cells is as high as in living organisms. Therefore, it has a cell layer barrier function similar to that found in living organisms. According to the method of the present invention, a cell sheet with functions similar to those in living organisms can be obtained can be obtained.

In the cell sheet obtained by the method of the present invention, a tight junction is formed between retinal pigment epithelial cells, and a basement membrane is formed on a contact face with the collagen gel. Here, the "basement membrane" is a membrane formed from the components produced from retinal pigment epithelial cells, and means a membrane that contains at least a part of the basement membrane component (hereinafter referred to as "basal membrane of retinal pigment epithelial cells"). The basal membrane of retinal pigment epithelial cells in living organisms is present as a thin film between a layer of retinal pigment epithelial cells and an inner collagen layer that constitutes the Bruch membrane, and is an extracellular matrix that has type collagen IV, laminin, heparan sulfate proteglycan (perlecan), nidogen and the like as representative components. The Bruch membrane is a thin film between the retinal pigment epithelial cell layer and the choroid, and has a 5-layer structure, namely a basal membrane of retinal pigment epithelial cells, an inner collagen layer, a layer of elastin, a layer of external collagen, and a basal membrane of choriocapillary lamina. The cell sheet contains a part (basal membrane of retinal pigment epithelial cells) of the Burch membrane structure. The formation of the tight junction can be confirmed by observing the shape of closely adhered cells with hexagonal shape, and the expression of ocludin, ZO-1 and the like between the cells by immunoblotting. Basal membrane formation can be confirmed by observing the expression of basal membrane markers, such as laminin, heparan sulfate proteoglycan (perlecan), nidogen, or type IV collagen and the like on a cell surface by immunoblotting, or electron microscopic observation of swept.

In general, retinal pigment epithelial cells cultured on a culture plate produce basement membrane components, but it is extremely difficult to detach the cells in the form of a sheet of usable retinal pigment epithelial cells detached from a culture plate (Invest. Ophtalmol Vis. Scie., 36 (2), 195, 381-390). In accordance with the method of the present invention, retinal pigment epithelial cells together with a basement membrane produced from retinal pigment epithelial cells can be recovered like a sheet without using an artificial membrane. Since retinal pigment epithelial cells form a monolayer structure, when handled individually, the sheet structure disintegrates and the cells spread into cell units. Thus, transplanting them as a sheet is extremely difficult. On the other hand, since the cell sheet is accompanied by a basement membrane and has sufficient rigidity, it does not wrinkle easily during recovery, which facilitates its handling extraordinarily. Consequently, as the assembly in a cell transplant device and a transplant operation can be performed smoothly, the cell transplant can be minimally invasive, and both the effect and the prognosis are expected to improve. In addition, since the cell sheet is accompanied by a basement membrane, it is extremely advantageous for transplantation in a disease in which the basement membrane is simultaneously discolored. For example, age-related macular degeneration is sometimes accompanied by abnormal Bruch's membrane. The basement membrane in the cell sheet of the present invention compensates for the altered part, whereby the graft rate of the cell sheet can be improved, and a treatment effect thereof can also be expected. Therefore, the cell sheet of the present invention is preferable as a transplant sheet that specifically targets a disease with an altered basement membrane, and can preferably be used as a transplant sheet that is particularly aimed at macular degeneration associated with age.

The method of producing the cell sheet may further comprise the following step (3):

(3) confirm the presence or absence of a basement membrane on the contact surface between the detached cell sheet and the collagen gel.

In step (3), the formation of a cell sheet having a cell layer composed of retinal pigment epithelial cells and a basement membrane can be determined by confirming the presence or absence of the basement membrane of the cell sheet. The presence or absence of the basement membrane can be confirmed by a method similar to the aforementioned confirmation of the formation of the basement membrane, for example, expression of a basement membrane marker, scanning electron microscope observation and the like. For the detection of the basement membrane, the expression of a basement membrane marker can be confirmed at any site in the cell (for example, the cytoplasm, the cell membrane, the nuclear membrane and the like). Preferably, the blank is a marker expressed on a contact face with the collagen gel.

The basement membrane marker herein includes a transcription product, a translation product or a degradation product in a gene specifically expressed in the basement membrane. Examples of such a gene include laminin, heparan sulfate proteglycan (perlecan), nidogen, type IV collagen and the like. Of these, laminin, type IV collagen and the like are preferably used, which are major components of the basement membrane.

A sample to be used to "confirm the presence or absence of a basement membrane on the contact surface between the detached cell sheet and the collagen gel" is not particularly limited as long as it contains a basement membrane marker (for example , RNA, protein, degradation product thereof and the like) obtained from the cell sheet (or cell) detached in step (2).

The expression of the basement membrane marker gene when the aforementioned sample is RNA can be examined by preparing a fraction of RNA (eg, total RNA, mRNA) from the cell of the cell sheet detached in step (2) and detecting a transcription product of the marker gene contained in the fraction, or directly detecting a marker gene product in the cell without extracting RNA from the cell.

When a fraction of RNA (eg, total RNA, mRNA) is prepared from the cell, it can be prepared using a known method, such as the guanidine-CsCl ultracentrifugation method, the AGPC extraction method and the like. Using a commercially available RNA extraction kit (for example, RNeasy Mini Kit; manufactured by Qiagen, etc.), total RNA with high purity can be prepared quickly and conveniently from traces of a sample. Examples of the method for detecting a transcription product of a basement membrane marker gene in an RNA fraction include a method that uses hybridization (Northern blot, stain transfer, DNA chip analysis, etc.), a method that uses PCR. (RT-PCR; competitive PCR, real-time PCR, etc.) and the like. Quantitative PCR methods such as competitive PCR, real-time PCR and the like are preferred, since the expression variation of a basement membrane marker gene can be detected quickly and conveniently from traces of a sample, and a chip analysis DNA is preferable since the expression variation of plural marker genes can be detected collectively and the quantification performance can also be improved by selecting a detection method and the like.

When Northern blot or stain transfer hybridization is used, the basement membrane marker gene can be detected using a nucleic acid (probe) capable of hybridizing with a gene transcription product. Examples of such acid include a nucleic acid capable of hybridizing with a transcription product of a basement membrane marker gene under conditions of high stringency. Examples of "high stringency conditions" include hybridization reaction at 45 ° C in 6xSSC (sodium chloride / sodium citrate), followed by washing one or more times at 65 ° C in 0.2xSSC / 0.1% SDS and the like The person skilled in the art can easily establish a desired stringency by appropriately changing the salt concentration of a hybridization solution, the hybridization reaction temperature, the probe concentration, the length of probe, number of discrepancies, hybridization reaction time, concentration of wash salt, wash temperature and the like. The nucleic acid can be DNA, RNA or DNA / RNA chimera, giving preference to DNA.

The nucleic acid to be used as a probe can be double stranded or single stranded. When it is double stranded, it can be double stranded DNA, double stranded RNA or a DNA: RNA hybrid. When it is single stranded, an antisense strand can be used. Although the length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target nucleic acid, it has, for example, not less than about 15 bases, preferably not less than about 30 bases. To allow detection and quantification of the target nucleic acid, preferably the nucleic acid is labeled. Examples of the label include a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like. Examples of radioisotopes include [32P], [3H], [14C] and the like. As the enzyme, a stable enzyme having a high specific activity is preferable, for example, p-galactosidase, p-glucosidase, alkaline phosphatase, peroxidase, malic acid dehydrogenase and the like. Examples of fluorescent substance include fluorescamine, fluorescein isothiocyanate and the like. Examples of luminescent substance include luminol, obtained from luminol, luciferin, lucigenin and the like. In addition, biotin- (strept) avidin can also be used to attach a probe and a tag.

When a Northern hybridization is used, an RNA fraction prepared as mentioned above is separated by gel electrophoresis, transferred to a nitrocellulose membrane, nylon, polyvinylidene difluoride and the like, hybridized under the "high stringency conditions" mentioned above in a hybridization buffer containing a labeling probe as mentioned above, and the amount of the membrane bound label for each band is measured by a suitable method, and thus the expression level of each marker gene can be measured of basement membrane. Also in the case of stain transfer, a stained membrane with an RNA fraction undergoes a similar hybridization reaction (performed for each marker gene), and the amount of the label in the stain is measured, and thus the measurement can be measured. expression level of each marker gene. When DNA chip analysis is used, for example, a cDNA is synthesized into which a suitable promoter, such as T7 promoter and the like, has been introduced, by reverse transcription reaction, from an RNA fraction prepared as has been mentioned above, cRNA is synthesized using RNA polymerase (in this case, that obtains the cRNa labeled using a biotin-labeled mononucleotide and the like as a substrate). The labeled cRNA is contacted with a chip that has the aforementioned probe immobilized thereon to perform a hybridization reaction, and the amount of the label bound to each probe on the solid phase is measured, and thus the level can be measured of expression of each basement membrane marker gene. This method is advantageous in terms of speed and convenience as the number of differentiated marker genes detected increases (therefore, probes to be in solid phase).

On the other hand, when a marker gene is detected without extracting RNA from the cell, in situ hybridization can be used as a means of detection. In this method, the cell is immobilized by treating it with a fixing agent, preferably, a precipitation fixing agent, for example, acetone, or incubating it for a short period of time in a solution of pH regulating formaldehyde, instead of extracting RNA from the cell. After immobilization, the cell is embedded in paraffin to form a block, and a cut slice thereof can be used as a sample. A well-prepared paraffin embedded sample can be stored at room temperature for many years. As the nucleic acid to be used as a probe, any one similar to the aforementioned examples can be used. Preferably, in situ hybridization is used in the present invention, since the expression of a basement membrane marker on the contact surface between the cell and the collagen gel can be directly confirmed.

Alternatively, the expression of a basement membrane marker in the sheet of cells separated in step (2) can be confirmed by preparing a protein fraction of the cell sheet (or cell), and detecting a translation product (i.e., marker protein) of the marker gene contained in the fraction, or by directly detecting a translation product of the marker gene in the cell sheet (or cell), without extracting the protein from the cell sheet (or cell). A marker protein can be detected by an immunological measurement method (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody for each protein and, in the case of a protein that shows a measurable physiological activity, such as An enzyme and the like can be detected by measuring the physiological activity of each marker protein by a known method. Alternatively, a marker protein can also be detected by a mass spectrometry method, such as MALDI-TOFMS and the like.

An antibody can be obtained for each marker protein according to a technique of producing monoclonal antibodies or polyclonal antibodies generally used and using a marker protein or protein, or a partial peptide thereof as an immunization antigen.

When respective immunological measurement methods are applied to the test method of the present invention, it is not necessary to establish special conditions or operations, and the like. A basal membrane marker protein measurement system can be constructed by adding the general technical considerations of the person skilled in the art to general conditions and operating methods in each method. As for the detail of these general technical means, compendiums, books and the like can be mentioned. For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "cont. Radioimmunoassay ”edited by Hiroshi Irie (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Igaku-Shoin, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Igaku-Shoin, published in 1987), "Methods in Enzymology", vol. 70 (Immunochemical Techniques (Part A)), ibidem, Vol. 73 (Immunochemical Techniques (Part B)), ibidem, Vol. 74 (Immunochemical Techniques (Part C)), ibidem, Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibidem, Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibidem, Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by the Academic Press) and the like.

Also described is a cell sheet obtained in accordance with the above-mentioned cell sheet production method, preferably, a cell sheet for transplantation comprising a cell layer formed from retinal pigment epithelial cells obtained by induction of ex vivo differentiation, and a basement membrane The retinal pigment epithelial cell sheet of the present invention is preferable as a transplant material for the retinal treatment of patients with ophthalmic diseases. Examples of ophthalmic diseases include retinal degenerative diseases, such as age-related macular degeneration disease, retinitis pigmentosa, diabetic retinopathy, retinal detachment and the like. Since the cell sheet of the present invention contains a basement membrane, it can be transplanted with a high graft rate for a disease that simultaneously involves an alteration of the Bruch membrane. In addition, since the retinal pigment epithelial cell sheet of the present invention has a basement membrane made from components similar to those found in living organisms, it can also be used for various screening purposes, such as efficacy screening. , toxicity assessment and the like in ophthalmic diseases mentioned above. For the screening of efficacy for the aforementioned ophthalmic diseases, for example, the cell sheet can be applied to the screening of a substance that is effective for the aforementioned ophthalmic diseases, according to the method described in JP-A-2007-500509. To be specific, the cell sheet of the present invention is cultured in the presence or absence of a candidate substance that is effective under the stress conditions possibly causing the aforementioned ophthalmic diseases (e.g., light (e.g., white light, blue light; light induces death of retinal cells, in particular photo-receptor cells, and may be an inciting factor of macular degeneration), A2E [N-retinylidene-N-retinyl-retinoid ethanolamine] (the accumulation of A2E is considered contributes to neurodegeneration associated with the age of retinal cells, in particular to the expression of macular degeneration), cumulative smoking time (smoking is considered a risk factor for macular degeneration), external pressure (for example, pressure hydrostatic; it is suspected that the increase in intraocular pressure is involved in glaucoma)), and the evaluation can be performed based on the number d and photoreceptors expressing rhodopsin, and by immunoblotting using the anti-caspase 3 antibody. For toxicity assessment, the cell sheet can be applied for screening for a toxic substance according to the method described in JP-A-2007-517210. To be specific, the cell sheet of the present invention is cultured in the presence or absence of a candidate substance to be toxic and using the integrin marker peptide described in JP-A-2007-517210, excited with a laser at a wavelength 488 nm, and fluorescence at 520 nm is detected for evaluation. Moreover, the retinal pigment epithelial cell sheet can also be used as an in vitro model for the evaluation of various in vivo functions of retinal pigment epithelial cells, such as the function that refers to the maintenance of visual cells, such as phagocytic capacity of the outer segment of photo-receptors, neuroprotective action and the like, barrier function of retinal blood vessels such as pumping action, tight junction and the like.

The cell sheet for transplantation can be used for the treatment of the aforementioned diseases in humans and mammals other than humans (e.g., monkey, mouse, rat, dog, bovine, horse, pig, sheep, goat, cat, rabbit, hamster , guinea pig, etc.).

The range of the disease area to which the cell sheet can be applied for transplantation is appropriately determined depending on the target disease, the animal species, age, sex, body weight and the symptoms of the administration subject, and the like.

The cell sheet for transplantation can be transplanted once or several times. The number of transplant applications is determined by health care professionals according to the disease and guidelines. For example, when the disease is age-related macular degeneration, the cell sheet for transplantation can be transplanted two or more times depending on the severity of the disease. When the transplant is performed several times, the interval is not particularly limited, and a period of several days to several weeks can be established.

The cell sheet for transplantation is transplanted by health care professionals according to an appropriate transplant method according to the guidelines. When the transplant cell sheet of the present invention is transplanted under the retina, a transplant method may be employed that includes delivering the sheet over a flow of water from a perforated injection needle, to the transplantation site under the retina of the eyeball , or an exclusive therapeutic apparatus for transplantation can also be used.

The present invention is explained in greater detail below with reference to Examples, which are mere exemplifications and do not limit the scope of the present invention in any way.

Production example 1 . Preparation of retinal pigment epithelial cells

As epithelial retinal pigment cells for use in obtaining the sheet in Example 1 below, mature retinal pigment epithelial cells (253G1, K11PD2, 59M8, 59SV2, 59SV3, 59SV9, 46a, K21EV15, 101EV3, K11EV9, 454E2 were used ) obtained by inducing differentiation of iPS cells, and retinal pigment epithelial cells (hES, CMK6) obtained by inducing differentiation of ES cells, according to the method described in Neuroscience Letters 458 (2009) 126-131.

Epithelial retinal pigment cells derived from human iPS

253G1 is a retinal pigment epithelial cell obtained by inducing differentiation of human iPS cells obtained from a healthy human being, and K11PD2 and 59M8 are retinal pigment epithelial cells induced to differentiate from human iPS cells obtained from patients with different retinitis pigmentosa. each. IPS cells were established by a method that includes introducing Oct3 / 4, Sox2, Klf4 and c-Myc genes into fibroblasts obtained from human skin using retroviruses, according to the method described in Cell 131, 861-872, 2007.

59SV2, 59SV3 and 59SV9 are retinal pigment epithelial cells obtained by inducing differentiation of human iPS cells obtained from the same retinitis pigmentosa patient. The iPS cells were established by a method that includes introducing Oct3 / 4, Sopx2, Klf4 and c-Myc in fibroblasts obtained from human skin using Sendai virus, according to the method described in Proc. Jpn Acad., Ser. B 85 (2009) 348-362.

K21EV15, 101EV3, K11EV9 and 454E2 are retinal pigment epithelial cells obtained by inducing differentiation of human iPS cells obtained from patients with different retinitis pigmentosa. The iPS cells were established by a method that includes introducing Oct3 / 4, Sox, 2, Klf4, L-Myc and LIN28 in human fibroblasts obtained from human skin using episomal vector, according to the method described in Nat Methods. 2011 May; 8 (5): 409-12).

Epithelial retinal pigment cells obtained from monkey iPS

46a is a retinal pigment epithelial cell obtained by inducing differentiation of an iPS monkey cell (mono cinomolgo), according to the method described in Jpn. J. Transplant. 44, 231-235.

Epithelial cells of retinal pigment obtained from ES

hES is a retinal pigment epithelial cell obtained by inducing differentiation of the human ES cell line khES-1. c MK6 is a retinal pigment epithelial cell obtained by inducing differentiation of monkey ES cells, according to the method described in Neuroscience Letters 458 (2009) 126-131.

Example 1. Production method of retinal pigment epithelial cell sheet

Preparation of mixed collagen gel solution

The following were prepared: A: Acid soluble type I collagen, obtained from Cellmatrix IA pig tendon (Nitta Gelatin, 3.0 mg / ml), B: culture medium concentrated at a concentration of 5 times [DMEM / F12 (Invitrogen, 12500-062, 3 g) was dissolved in MilliQ water, and the total volume (50 ml) was treated with filter], and C: reconstitution buffer [1N NaOH (50 mM; 5 ml), NaHCO ³ (260 mM, 2.2 g) and HEp Es (200 mM, 4.77 g) were dissolved in MilliQ water, and the total volume (100 ml) was treated by filtration]. Under cooling, B (2 vol) was mixed (pale yellow) with A (7 vol) without bubbling. Then, C (1 vol) was added and the mixture was stirred (pale pink) to give a mixed solution of 0.21% collagen gel.

Preparation of retinal pigment epithelial cell sheet

The mixed solution of 0.21% collagen gel (200 pl) was added to the insert of a 12 mm Transwell insert (polyester membrane with pores of 0.4 pm); Cornig, 3460), and the mixture was incubated at 37 ° C for 30 min. Then, F10-10% Fb S [F-10 (Sigma, N6908, 445 ml), FbS (50 ml), penicillin-streptomycin (Invitrogen, 15140-122.5 ml)] was added in an amount of 1500 pl at outside of the insert and 500 pl inside the insert, and the Transwell membrane was incubated at 37 ° C for 24 h. Subsequently, the inside and outside of the insert were washed once with F10-10% FBS, epithelial cells of respective retinal pigment obtained in Production Example 1 to 5x105 were seeded ^(f 10-109% FBS, 500 pl) in the insert, and F10-10% F ^b S (1500 pl) was added to the outside of the insert. Retinal pigment epithelial cells were cultured in F10-10% FBS until confluence. After reaching the confluence, the medium was changed to SFRM-B27 [DMEM (Sigma, D6046, 350 ml), F12 HAM (Sigma, N6658, 150 ml), B27 (Invitrogen, 17504-044, 10 ml), 200 mM L-glutamine (Sigma, G7513, 5 ml), penicillinestreptomycin (Invitrogen, 15140-122, 5 ml), bFGF (wako, 060-04543, 10 ng / ml)] (1500 pl outside the insert 500 pl inside the insert, the change of medium was made 3 times / week), and the retinal pigment epithelial cells were cultured until they showed adequate color and shape.

cut

After progressing for 6 weeks from the start of the culture, the membrane of the insert was separated, collagenase L (Nitta Gelatin, PBS (+): Sigma, 2600 U / ml, 100 jl) was added under the insert, and the insert was incubated at 37 ° C for 60 min and washed 3 times with PBS (+). SFRM-B27 was added in the form of drops so that the retinal pigment epithelial cell sheet did not dry out and was cut to the desired size with PALM MicroBeam (ZEISS).

Example 2. Production method of retinal pigment epithelial cell sheet (type of collagen) Cell sheets were produced and cut to give retinal pigment epithelial cell sheets in the same manner as in Example 1 except that, in the step of producing a cell sheet using 253G1 (iPS retinal pigment epithelial cells) in Example 1, (A) type I collagen TE obtained from pig skin was used (special order product: containing mainly type I collagen , a small amount of collagen type lIl, Nitta Gelatin, 5 mg / ml) as a mixed solution of 0.35% collagen / well, (B) type I collagen T-1002 obtained from pig tendon (product) was used Special order: type I collagen, Nitta Gelatin, 5.1 mg / ml) as a mixed 0.35% collagen solution / well, (C) FITC I labeled collagen (Chondrex, 1 mg / ml) was used as 0.07% collagen mixed solution / well, (D) FITC labeled collagen I (special order, Chondrex, 3 mg / ml) was used as a mixed 0.21% collagen solution / well, (E) atelocollagen (KOKEN, 3 mg / ml) was used as a mixed collagen solution at 0., 215% / well, and (F) permeability collagen membrane was used for cell culture (KOKEN), respectively, instead of acid soluble Cellmatrix IA type I collagen obtained from pig tendon (Nitta Gelatin , 3 mg / ml) as a mixed solution of 0.21% collagen / well.

The test results of Example 1 and the cases using each of the aforementioned collagens were compared and evaluated in terms of 4 properties [1. Gel strength; 2. Cell adhesion; 3. Cell growth; 4. Security]. The results were, (A) {1. Lower; 2. Equivalent; 3. Bottom; 4. Good}, (B) {1. Good (5.1 mg / ml); 2. Equivalent; 3. Bottom; 4. Good}, (C) {1. Lower (1 mg / ml); 2. Bottom; 3. Unknown; 4. Unknown}, (D) {1. Equivalent (3 mg / ml); 2. Equivalent; 3. Bottom; 4. Unknown}, (E) {1. Equivalent (3 mg / ml); 2. Bottom; 3. Unknown; 4. Good}, and (F) {was not lysed by collagenase, so it was unusable}. As for gel strength, a certain level of strength is required to allow the growth of retinal pigment epithelial cells. From this aspect, the particularly preferable types and concentration of collagen were the acid-soluble Cellmatrix IA type I collagen obtained from the pig tendon of Example 1 and (B) the T-1002 type I collagen obtained from the tendon. of pork used at the aforementioned concentration. When the substrate does not have a certain level of strength, the retinal pigment epithelium does not grow and cannot be used for the present invention.

Example 3. Method of producing retinal pigment epithelial cell sheet (amount of collagen) Cell sheets were produced and cut in the same manner as in Example 1 except that, in the stage of producing a cell sheet using 253G1 (cells epithelial retinal pigment of iPS) of Example 1, the amount of mixed collagen gel solution to be used was changed from 200 µl to 100 µl to 300 µl, so sheets of epithelial retinal pigment cells were recovered.

Compared to Example 1, when the amount of collagen gel mixed solution used was 100 µl, a thin collagen gel layer formed in the central part due to an influence of surface tension caused by the small amount of the mixed solution of collagen gel and, as the culture proceeded, seeded retinal pigment epithelial cells directly contacted the bottom membrane easily, which caused rupture of the retinal pigment epithelial cell sheet during the cutting operation of the sheet. When the amount of collagen gel mixed solution used was 300 µl, as the amount of collagen gel mixed solution was high, a thick collagen gel layer formed, which relatively reduced the amount of medium that could be retained. at the insertion, and therefore, the maintenance of the culture was not easy to perform, the collagenase treatment took time and it was feared that the damage to the cell sheet would become larger. When the amount of collagen gel mixed solution used was 100 µl, the cells came into direct contact with the membrane, and the cell sheet was broken by that part when the membrane was removed.

Example 4. Production method of retinal pigment epithelial cell sheet (amount of collagenase and treatment time)

The cell sheets were produced and cut in the same manner as in Example 1, except that in the step of producing a cell sheet using 253G1 (iPS retinal pigment epithelial cell) of Example 1, 1% of Collagenase L (Nitta Gelatin) or collagenase type I (Roche) with the retinal pigment epithelial cell sheet for 10 min in an amount of 10 jl, 20 min in an amount of 10 jl, 30 min in an amount of 10 jl, 20 min in an amount of 20 jl, 60 min in an amount of 20 jl, and 50 min in an amount of 30 jl, instead of 30 min in an amount of 30 jl, so that sheets of epithelial cells were recovered from retinal pigment

As a result, when a collagenase treatment was performed for 60 min in an amount of 10 ml, or 60 min in an amount of 20 µl, collagen degradation was observed at the same level as with 30 µl for 30 min. Example 5. Production method of retinal pigment epithelial cell sheet (number of seeded cells)

Cell sheets were produced and cut in the same manner as in Example 1 except that, in the step of producing a cell sheet using 253G1 (iPS retinal pigment epithelial cell) of Example 1, the number of cells that were going to be planted inside the insert, 5x105 cells / 500 jla (A) 5x104 cells / 500 jl, (B) 1x105 / 500 jlo (C) 1x106 / 500 jl were changed, so sheets of epithelial cells of retinal pigment were recovered.

Compared to Example 1, (A) and (B) required a longer time to reach cell confluence due to the small number of cells, and (C) showed slow growth and also tended to require a longer time to reach the cellular confluence

Example 6. Basal membrane formed on retinal pigment epithelial cell sheet

A cryosection (frozen section) was prepared from the cell sheet produced from 253G1 (iPS retinal pigment epithelial cell) in Example 1 and subjected to immunohistochemical staining. The formation of a tight junction was confirmed by the expression of ZO-1, and the formation of a basement membrane was confirmed by the expression of laminin and type IV collagen. For the detection of each protein, the respective anti-ZO-1 rabbit antibodies manufactured by Zymed (1: 100 dilution), rabbit laminin manufactured by Abcam (1: 200 dilution) and anti-human type IV collagen antibody were used mouse manufactured by Calbiochem (1:40). In addition, it was confirmed that the retinal pigment epithelial cell sheet has a single layer epithelial form from the nuclear spot state using 4 ', 6-diamidino-2-phenylindole manufactured by Molecular Probes (DAPI; 1 jg / ml) .

Evaluation 1. Extinction profile of specific epithelial cell retinal pigment gene

In the step of producing a 59SV3, 59SV9 (iPS retinal pigment epithelial cell) cell of Example 1, the expression of BEST1, RPE65, MERTK, CRALBP in the cells that constituted the sheets after the course of 1 week, 4 weeks, 2 months, where the day on which the medium was changed to SFRM-B27 after cell confluence was day 0, was confirmed by RT-PCR. As a result, expression was observed at the same level as that of the positive control (total RNA of human retinal pigment epithelial cells (manufactured by ScienCell, Cat. No. 6545)). Here, BEST 1, RPE65, MERTK are genes specifically expressed in retinal pigment epithelial cells. CRALBP is a gene expressed in retinal pigment epithelial cells and Muller cells.

Evaluation 2. Measurement of residual collagen in retinal pigment epithelial sheet

Cryosections (frozen sections) were prepared by cutting, before and after collagenase treatment, from respective cell sheets produced from 253G1 (iPS retinal pigment epithelial cell) in Example 1, and subjected to immunohistochemical staining. The nucleus was stained with 4 ', 6-diamidino-2-phenylindole (DAPI; 1 jg / ml) manufactured by Molecular Probes, and type 1 collagen was stained with rabbit anti (human type I collagen) antibody (dilution 1 : 40) manufactured by Calbiochem. As a result, no collagen was detected in the sheets after collagenase treatment, and it was confirmed that the collagenase removed the collagen that covered the culture plate. On the other hand, collagen was detected in the cut sheets before collagenase treatment.

Evaluation 3. Cytokine secretion capacity of retinal pigment epithelial cell sheet

Culture media on the Apical side and the Basal side in the transwell membrane were recovered before the step of cutting retinal pigment epithelial cell sheets in cell sheets produced from 253G1 (iPS retinal pigment epithelial cell) and 454E2 (iPS retinal pigment eptielial cell) of Example 1, and the production quantities of VEGF and PEDF were detected by ELISA according to the method described in Arvydas M, IOVS, 2006; 47: 3612-3624. As a result, it was confirmed that, similar to the retinal pigment epithelium obtained from human embryo disclosed in Arvydas M, IOVS. 2006; 47: 3612-3624, VEGF was secreted mainly on the Basal side, and PEDF was secreted mainly on the Apical side (Fig. 1). It was shown that the cell sheet of the present invention has a secretory capacity of cytokine similar to that produced in living organisms, and is superior in functionality.

Evaluation 4. Transepithelial electrical resistance of the retinal pigment epithelial cell sheet

A strong correlation is seen between the barrier function of a cell layer and the impedance, namely transepithelial / transendothelial electrical resistance (TER). A probe was placed in the interior and exterior of the insert according to the method described by MILLIPORE (using Millicell ERS-2), before the step of cutting the retinal pigment epithelial cell sheet produced from 454E2 (epithelial cell of iPS retinal pigment) in Example 1, and the TER was measured electrically. As a result, TER was 640ü cm2, and showed a TER value high as the retinal pigment epithelium obtained from human embryo disclosed in Nature Protocols vol 4, No. 5662-673 (2009), Fig. 10. The cell sheet of the present invention was shown to have a high function of barrier similar to that of living organisms.

Evaluation 5. Transplantation of retinal pigment epithelial cell plate obtained from monkey ES cells

A sheet of monkey retinal pigment epithelial cells produced from retinal pigment epithelial cells obtained from monkey ES cells, CMK6 in Example 1, was transplanted into an eye of a monkey according to the method described in Invest Ophthalmol Vis Sci. 1995 Feb; 36 (2): 381-90. Before the transplant, a retinal photocoagulation was performed to alter the retina of the eye that was going to undergo the transplant. On day 28 of the transplant in the eye of the monkey that had the retinal photocoagulation macula formed in it, photographs of the fundus were taken, and images of the ocular fundus sections were generated as histological sections using OCT (optical coherence tomography ), and the condition of the retina was confirmed based on them. As a result, fluorescence leakage was not found by fluorescein angiography, the graft survived and no alterations such as thinning of the retina and the like were found.

Evaluation 6. Transplantation of retinal pigment epithelial cell plate obtained from monkey iPS cells

A sheet of monkey retinal pigment epithelial cells produced from retinal pigment epithelial cells obtained from monkey iPS cells, 46a in Example 1, was transplanted under the retina of one eye for autologous transplantation and three eyes for transplantation crossed according to the method described in Invest Ophthalmol Vis Sci. 1995 Feb; 36 (2). 381-90. Up to one year after the transplant, photographs of the fundus were taken and images of the ocular fundus sections were produced as histological sections using OCT (optical coherence tomography), based on which the condition of the retina was observed with the over time. In cross-transplants, clear rejection reactions were found such as fibrous changes on the periphery of the graft, fluorescence leakage by fluorescein angiography and high-gloss lesion under the retina by OCT. On the other hand, in the autologous transplant, no such obvious rejection was observed, nor was fluorescence leakage by fluorescein angiography, the graft survived and no alterations such as sensory retinal thinning and the like were found.

Industrial applicability

Using the method of the present invention, a retinal pigment epithelial cell sheet can be comparatively produced to be applied by transplantation to patients with age-related macular degeneration. In particular, when the cells to be used for culture are retinal pigment epithelial cells obtained from iPS, the patient's own cells can be used, which can prevent transplant rejection. Moreover, since the cell sheet obtained by the method of the present invention has a basement membrane made from the same components as in living organisms, a retinal pigment epithelial cell sheet more similar to the state in living organisms can be reproduced, which is useful for various screening purposes.

Claims (5)

1. A method for producing a cell sheet composed of retinal pigment epithelial cells, a tight junction formed between retinal pigment epithelial cells, and a basement membrane formed on a contact face with the collagen gel, comprising the following steps (1) sowing and culturing retinal pigment epithelial cells on a collagen gel to form a cell sheet composed of retinal pigment epithelial cells, the tight junction formed between retinal pigment epithelial cells, and the basement membrane formed on one face contact with the collagen gel, and (2) degrade the collagen gel with collagenase to detach the cell sheet composed of retinal pigment epithelial cells, the tight junction formed between the retinal pigment epithelial cells, and the formed basement membrane on a contact face with the collagen gel. 2. The method of producing the cell sheet according to claim 1, wherein the retinal pigment epithelial cells are cells obtained by inducing differentiation of stem cells or progenitor cells. 3. The method of producing the cell sheet according to claim 2, wherein the stem cells are ES cells or iPS cells. 4. The method of producing the cell sheet according to claim 1, wherein the concentration of collagen in the collagen gel is 0.1% -0.5%. 5. The method of producing the cell sheet according to any one of claims 1 to 4, further comprising the following step (3): (3) confirm the presence or absence of a basement membrane on the contact surface between the detached cell sheet and the collagen gel.